Solvent extraction process

ABSTRACT

The present invention relates to an improved extraction process for an alkaloid glycoside extract from the species  Solanum . In this process, a ground, dried plant tissue of the species  Solanum  is subjected to alcohol extraction using methanol, the extract is dried and dissolved in a weak volatile acid and centrifuged, the supernatant is precipitated using a volatile base, and the precipitate is thoroughly washed and dried. Extracts having high purity, and products made by the process are also provided.

[0001] The present invention relates to an improved extraction processfor an alkaloid glycoside extract from the species Solanum.

[0002] A standard mixture of solasonine glycosides, isolated from thefruits of Solanum sodomaem, is known as BEC. BEC is a crude mixture ofglycoalkaloids comprising the triglycerides solasonine (approx. 33%),solamargine (33%) and an undefined fraction which has been referred toas “di and monogylcosides”.

[0003] The process for making BEC involves homogenizing the fruits ofSolanum sodomaem in a large volume of acetic acid, filtering off theliquor through muslin, precipitating the glycosides with ammonia andrepeating the steps several times (WO 00/6 11 53; Planta Medica 1987, 1:59-62). The yield of the solasodine glycoside mixture is very low(approx. 0.8%).

[0004] BEC is incorporated in very small amounts into a topical cream(Curaderm®) which is used to treat skin lesions such as sunspots andkeratosis.

[0005] BEC has also been reported to exhibit preferential cytotoxicityfor various human cancer cells (WO 91/10743).

[0006] While BEC is of sufficient quality to be used in small quantitiesin over the counter (OTC) topical cream formulations, it does not meetthe regulatory requirements for pharmaceutical use. Thus, the individualprocess steps of the standard process for the preparation of BEC are notdefined to GMP (Good Manufacturing Process) in terms of scale up,definition of yield, composition and product quality.

[0007] Moreover, other than the presence of the triglycosides solasonineand solamargine the BEC mixture has never been analyzed regarding theprecise nature of the active ingredients, the assumption being that allglycoside components are active in combination.

[0008] The present invention is directed towards a novel solanumglycoside extract comprising the active glycoside components in highpurity meeting the requirements of GMP.

[0009] The present invention thus provides a novel extraction processfor the preparation of a solanum glycoside extract consistingessentially only of the active solanum glycosides.

[0010] Also provided is a novel very sensitive HPLC method for theanalysis of solanum glycoside extracts. This novel HPLC method employsan ACE 5 C18 (V99-140) column (length 25 cm+1 cm guard cartridge;particle size 5 μm; internal diameter 4.6 mm; pore size 100 Å), AdvancedChromatography Technologies (ACT) (obtainable from HiChrom, Theale).

[0011] The mobile phase used at a flow rate of 0.7 ml/min was 75% 20 mM(pH 2.95) phosphate buffer, 25% acetonitrile using a Spectra-Physics SP8800 Ternary HPLC Pump. Detection was carried out at 205 nm with aSpectra-Physics, Spectra 1000 Variable Wavelength Detector.

[0012] Based on extensive analysis of the BEC mixture it was shown thatin addition to the triglycosides solasonine and solamargine adiglycoside component is present in an amount of approximately 30%. Itwas surprisingly found that the diglyceride component does not appear toexhibit any activity in cytotoxicity in various experimental cancer celllines.

[0013] In accordance with the present invention a solasodine glycosideextract is provided consisting essentially of the triglyceridessolasonine and solamargine.

[0014] Preferably the extract is of close to 100% purity with nomeasurable or only trace amounts of the inactive diglycoside component.

[0015] Preferably, solasonine is present in the final extract in anamount between 30-50% whereas solamargine is present in an amount of40-60%.

[0016] The extract of the present invention may be obtained by thefollowing process:

[0017] The solanum plant tissue is dried and comminuted to a powder. Thepowder is subjected to an alcohol extraction. Following the removal ofthe alcohol the dried extract is dissolved in acid and centrifuged. Thesupernatant is precipitated under alkaline conditions. The acid/alkalineprecipitation steps may be repeated several times.

[0018] The precipitate is then thouroughly washed with water and driedand further purified by silica gel chromatography.

[0019] As a solanum glycoside source any of the plants of the Solanumspecies may be used. Preferably, however, the extract of the inventionis obtained from Solanum sodomaem. It has been found that the highestyields of the two components of the extract solamargine and solasoninemay be obtained from the lyophilized fruit of Solanum sodomaem.

[0020] While the extraction process may be carried out using anypharmaceutically acceptable volatile alcohol including methanol,ethanol, propanol and isopropanol, the extraction is most preferablycarried out using methanol.

[0021] For the precipitation step the dried extract may be treated withany volatile and weak acid and alkaline reagent. It has been found thatoptimal results may be obtained using acetic acid and concentratedammonia.

[0022] For the chromatographic purification using silica gelchromatography, the freeze dried extract may be dissolved in methanol.Before elution the silica gel is washed with acetone. The elution ispreferably carried out using a gradient of methanol and acetone.

[0023] In the subsequent example the extraction process of the inventionis exemplified in detail below.

EXAMPLE 1

[0024] 1.1 Preparation of Fruit

[0025] The fruits of Solanum Sodomaem were cut in half and dried bylyophilization. The dried fruits were then comminuted to a powder. Thefinal dried, ground powder from a yield of 5 kg of fruits was 898 g.

[0026] 1.2 Solvent Extraction of Glycoalkaloids

[0027] The powdered fruit (approx. 230 g each) was divided between 3Soxhlet thimbles (Whatman cellulose, 60×180 mm), and four extractionsconducted for each with 1 litre methanol (Labscan, HPLC grade). Thefirst extraction (1 litre methanol) was conducted for 7.5 hours beforethe methanol was removed by evaporation and the resulting extractsanalysed by HPLC. The 3 subsequent extractions were with 750 ml methanolto find the point at which all glycoalkaloids were completely extracted.The four extractions were labelled A, A1, A2 and A3, respectively. Theconcentrations of glycoalkaloid in the the four separate extractions areshown in Table 1. TABLE 1 Concentration of glycoalkaloids in the ExtractSolasonine, mg/ml Solamargine, mg/ml by HPLC by HPLC A  7.9 9.4 A₁ 2.22.6 A₂ 0.16 0.16 A₃ 0.06 0.07

[0028] 1.3.1. Production Scale Purification of Extract A

[0029] The dry and partially evaporated extracts A, A1 and A2 (A3abandoned due to lack of useful product) were pooled and evaporated toyield a pooled dry extract (62.23 g). The extracts were dissolved inacetic acid (2 litres, 3% (v/v)) and the slightly turbid solutionclarified by centrifugation (6,000 rpm, 20 mins). The supernatant wascollected and a small amount of green precipitate discarded. The samplewas then filtered to remove a few small remaining lumps of greenprecipitate.

[0030] 1.3.1.1 1^(st) Precipitation with Ammonia

[0031] The filtrate was adjusted to pH 9 by addition of concentratedammonia and the precipitate allowed to settle for 1 hour beforecentrifugation (6,000 rpm, 20 mins). This gave a gel-like precipitate,which was easily re-dissolved in approx. 1 litre 3% (v/v) acetic acid,such that the final volume of sample was 1.25 litres.

[0032] 1.3.1.2 2^(nd) Precipitation with Ammonia

[0033] The pH of the sample was adjusted to 9 by addition ofconcentrated ammonia and the precipitate was allowed to settle for a fewminutes before centrifugation (6,000 rpm, 20 mins).

[0034] 1.3.1.3 Washing of Precipitate

[0035] The precipitate was re-dispersed and thoroughly mixed withde-ionised water (6×250 ml), and collected by centrifugation. Thisprocedure was repeated once and the pellets were drained.

[0036] 1.3.1.4 Freeze-Drying of Product A

[0037] The pellets were dissolved in ammonium acetate, pH 4.5 (3% (v/v)acetic acid adjusted to pH 4.5 with concentrated ammonia to give asolution of ammonium acetate at pH 4.5). The final volume of solution atthis stage was 360 ml. The sample was polish filtered through a glassfibre filter (Whatman GF/B) prior to weighing into plastic containers(16 cm×16 cm×6 cm). The sample was then frozen and subsequentlyfreeze-dried.

[0038] The yield (by weight) of the extract was approx. 1.14% of theoriginal weight of the whole fruit, or 6.45% of the dried powderedfruit.

[0039] The composition of the freeze dried extract was determined byHPLC. It was found to contain approx. 33% solasonine, 40.1% solamarginebut no detectable traces of the diglycoside component.

[0040] 1.4 The Freeze Dried Extract A

[0041] Chromatographic Purification of Extract A

[0042] A column was packed with dry Silica Gel (406.6 g, columndimensions: 7.5 cm×18 cm). Freeze-dried Extract A (13.59 g) was weighedout and dissolved in methanol (250 ml). Silica gel (106 g) was added,and the slurry was dried to a fine powder by evaporation. The slurry wasthen applied to the top of the silica column and a layer of silver sandwas added (1 cm approx.). The column was washed with acetone (1 litre)before elution of the glycoalkaloids. The column was eluted at a flowrate of 1 litre/hr with a 4-step gradient of methanol:acetone asfollows:

[0043] Step 1: methanol (30% (v/v)):acetone (70% (v/v)), 2 litres

[0044] Step 2: methanol (40% (v/v)):acetone (60% (v/v)), 2 litres

[0045] Step 3: methanol (60% (v/v)):acetone (40% (v/v)), 1 litre

[0046] Step 4: methanol (100% (v/v) 1.5 litres

[0047] 1.4.1 Pooling of Fractions

[0048] Fractions (51×25 ml) were collected when step 1 of the gradientwas applied, in order to monitor where the solamargine and solasoninestarted to elute. After this, larger 500 ml fractions were collectedthroughout until after step 4. The progress of the purification wasmonitored be TLC. Fractions containing the desired end products(solamargine, solamargine plus solasonine, as well as the solasonine)were pooled. The fractions were dried by rotary evaporation andprocessed immediately as described below.

[0049] 1.4.2 Ammonia Precipitation

[0050] The dried pooled fractions were dissolved in acetic acid (900 ml,3% (v/v)) and adjusted to pH 9 by addition of concentrated ammonia. Theresulting precipitate was collected by centrifugation (6,000 rpm/20mins).

[0051] 1.4.3 Washing of Precipitates and Freeze-Drying

[0052] The resulting precipitate was washed three times with water,collected by centrifugation, and frozen at −20° C., prior tore-dissolution in acetic acid (1.6 litres, 0.1% (v/v)). The sample wasthen divided equally into 8 containers and freeze-dried.

[0053] 1.4.4 Yields and Purities of Final Purified Products

[0054] The freeze-dried products were weighed and stored desiccatedunder vacuum at +4° C. The samples were then analysed for purity(against a BEC standard) by HPLC. The results are presented in Table 2.TABLE 2 Yields and Purities of Final product HPLC Analysis % Yield *% %% Total by Sample G recovery Solasonine Solamargine HPLC A 8.49 88.040.5 55.3 97.5

[0055] 1.5 Chemical and Microbiological Analysis of Original BEC PlantExtract and Final Product Prepared in Accordance with the Invention

[0056] Samples of the prior art BEC product as well as a product inaccordance with the present invention were analysed for moisture contentand for microbial, pesticide and heavy metal content.

[0057] 1.5.1 Microbial Analysis

[0058] A sample of the prior art BEC product, together with the finalpurified product of the invention were sent for microbial analysis,moisture, methanol, ash and heavy metal analysis.

[0059] Results for these analyses are summarised in Table 3. TABLE 3Summary of analytical results for BEC plant material and Purifiedproduct A Test BEC product A Bacterial count >1500 per 50 mg <1 per 10mg Fungal Count >1500 per 50 mg <1 per 10 mg Residue on 6.7 0.4ignition, % Water, % m/m 1.99 5.66 Methanol, μg/g 350 <50 Heavy metals,10 <10 ppm as Pb Heavy Metals: Cd, mg/kg <0.02 Pb, mg/kg <0.10 Hg, mg/kg0.01 As, mg/kg 0.03

2. CONCLUSIONS

[0060] Solvent extraction using methanol has proven to be verysuccessful in the extraction of the selective triglycosides solasonineand solamargine with little or no contamination with the diglycoside.

[0061] In order to obtain the final high purity product the extract wassubjected to a silica gel chromatography. Preferably, in order tooptimize the recovery of solasonine, which has a low solubility inacetone, a graded eluent comprising methanol and acetone should beapplied.

[0062] It was shown that the process of the present inventioneffectively removes the third component which is present in BEC inamounts in excess of 30%.

[0063] Following evaporation of the solvent from the sample, a singlere-precipitation with ammonia and washing gives rise to a clean product,which can easily be freeze-dried from solution in aqueous ammoniumacetate. The volatile constituents are removed during the freeze-dryingprocess. It is preferable that the final purification step is carriedout within a 24 hour period in order to optimise the appearance of thefinal product.

[0064] 2.1 Product Purity

[0065] The process developed here gives a significantly improved productcompared with that obtained by the Cham process (2), in terms of itsappearance, purity, moisture, and heavy metal content. The finalfreeze-dried product was a granular off-white powder. The purity of theproduct was in the range 92.7-99.3% (HPLC analysis), compared with atypical purity of 65-70% obtained by the previous method. Moisturecontent was also lower (4.0-5.7% compared with 7% obtained previously),and heavy metal contamination was extremely low.

1. A process for the preparation of a glycoside extract comprising thefollowing steps: a) ground, dried plant tissue of the species Solanum issubjected to alcohol extraction using methanol; b) the extract obtainedis dried and dissolved in a weak volatile acid and centrifuged; c) thesupernatant is precipitated using a volatile base; d) the precipitate isthoroughly washed and dried.
 2. A process according to claim 1, whereinthe steps b) and c) are repeated several times.
 3. The process accordingto claim 1, wherein the volatile acid is acetic acid.
 4. The processaccording to claim 1, wherein the base is concentrated ammonia.
 5. Theprocess of claim 1, wherein the dried plant tissue is lyophilized fruitof Solanum sodomaem.
 6. A process for the isolation of an extract of thetriglycosides solasonine and solamargine in substantially pure formcomprising subjecting the extract as obtained according to claim 1 tosilica gel chromatography.
 7. The process according to claim 6, whereinthe silica gel chromatography step uses an eluent that is amethanol/acetone gradient.
 8. A solasodine triglycoside extractcomprising the triglycosides solasonine and solamargine having a purityof above 90%.
 9. The solasodine triglycoside extract of claim 8, whereinthe ratio of solasonine:solamargine is in the range of 0.3-0.7:0.4-0.8.10. The solasodine triglycosides extract of claim 8, wherein the ratioof solasonine:solamargine is in the range of 0.4-0.6:0.5-0.7.
 11. Thesolasodine triglycoside extract of claim 8, wherein the ratio ofsolasonine:solamargine is 0.7:0.5.
 12. The solasodine triglycosideextract prepared by the process of claim 7, wherein the extractcomprises Solasonine and Solamargine having a purity of above 90%.